Summary of Work: The first clinically approved anti-viral drug against HIV-1 infection is zidovudine (3'azido thymidine nucleoside, AZT). AZT completely blocks viral replication in normal human peripheral blood mononuclear cells and in vitro AZT-triphosphate (AZT-TP) inhibits purified HIV-1 reverse transcriptase with a ki of 20 nM. In the cell AZT-TP has little inhibitory effect on the nuclear DNA polymerases but selectively targets ands inhibits the mitochondrial DNA polymerase. This inhibition of the mitochondrial DNA polymerase has been documented with patients undergoing antiviral drug treatment. These patients undergoing AZT treatment develop a mitochondrial dysfunctional disease known as red ragged fiber disease. Red-ragged fiber disease usually is a genetic disease of the mitochondrial DNA resulting from point mutations in the mitochondrial DNA, possibly arising through DNA replication errors from the mitochondrial DNA polymerase. How the mitochondrial DNA polymerase makes point and deletions mutations and what structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to its inhibition patterns is poorly understood. In addition, the mode and effect of antiviral nucleotide analogs, such as AZT, on the inhibition and fidelity of mitochondrial DNA replication is poorly understood. To better understand the mechanism of mitochondrial DNA replication and mitochondrial toxicity of antiviral drugs we are analyzing the unique structural features of the mitochondrial DNA polymerase gamma. Based on the homology between the S. cerevisiae DNA polymerase gamma and the bacterial DNA polymerases, we have cloned the DNA polymerase gamma genes and cDNA from S. pombe, D. melanogaster and Homo Sapiens. The human mitochondrial DNA polymerase protein has been functionally overexpressed greater than 100 fold in insect cells by a recombinant baculovirus. The overexpressed protein has been purified to homogeneity and enzymatically characterized. An exonuclease deficient site specific mutant protein of the human DNA polymerase gamma has been made and overexpressed in baculovirus. Likewise, a dideoxy-resistant DNA polymerase gamma protein has been made by site specific mutagenesis and overexpressed. These wild type and mutant polymerases will be studied in vitro to emulate conditions occurring in mitochondria compromised by AZT. We have entered into a collaboration with Bill Lewis (Cincinnati) to study the overexpression of the wild type and mutant DNA polymerase gamma proteins in a mouse transgenic system which expresses the cDNA of interest in a tissue specific manner.